title: An EGFR/CD13 bispecific fusion protein and its enediyne-energized analog show potent antitumor activity.
abstract: Targeting to two or more objectives simultaneously has been pursued as a strategy to potentially increase the efficiency and selectivity of targeted drugs to certain cancers. In this study, an epidermal growth factor receptor (EGFR)/CD13-targeting, bispecific fusion protein ER(Fv)-LDP-NGR consisting of an anti-EGFR single-chain variable fragment (scFv), an apoprotein (LDP) of lidamycin (LDM), and a tri-CNGRC (Cys-Asn-Gly-Arg-Cys) peptide against CD13 was constructed, and then an enediyne-energized analog ER(Fv)-LDP-NGR-AE was generated by integration with an enediyne chomophore (AE) derived from LDM. The apoprotein LDP was used as a 'scaffold' to connect the scFv fragment and the tri-CNGRC peptide and also served as a specific 'carrier' for the extremely potent cytotoxic enediyne chromophore of LDM. Compared with its monospecific counterparts, ER(Fv)-LDP and LDP-NGR, the bispecific fusion protein ER(Fv)-LDP-NGR showed higher affinity to EGFR/CD13-overexpressed tumor cells. Determined by the MTT assay, the bispecific, enediyne-energized ER(Fv)-LDP-NGR-AE showed highly potent cytotoxicity to EGFR/CD13-overexpressed MCF-7 cells, with an IC50 value of 3.4×10 mol/l, whereas for the EGFR-overexpressed A431 cells, the IC50 value was 2.2×10 mol/l. For MCF-7 cells, the bispecific ER(Fv)-LDP-NGR-AE was more potent in cytotoxicity than the corresponding monospecific energized fusion proteins. In athymic mice models, the bispecific fusion protein ER(Fv)-LDP-NGR presented stronger inhibitory activity than the monospecific ER(Fv)-LDP and LDP-NGR. For the enediyne-energized fusion proteins, ER(Fv)-LDP-NGR-AE significantly inhibited the growth of EGFR/CD13-overexpressed MCF-7 xenograft and EGFR-overexpressed A431 xenograft by 86.3 and 81.4%, respectively. In addition, the bispecific ER(Fv)-LDP-NGR-AE showed much higher efficacy than its monospecific analogs ER(Fv)-LDP-AE and LDP-NGR-AE in both MCF-7 and A431 xenograft models. The results show that EGFR/CD13 bitargeting effectively improved the antitumor efficacy. Both the bispecific fusion protein and its enediyne-energized analog are highly effective in athymic mice bearing xenografts, and the latter exerts more marked efficacy. Generation of a pair of bispecific antibody-based therapeutics and its corresponding antibody-drug conjugate simultaneously may be a feasible strategy for the development of new targeted drugs for cancer therapy.
authors.affiliation: Department of Tumor, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, People's Republic of China.
authors.forename: Weijin
authors.initials: W
authors.lastname: Sheng
authors.forename: Yue
authors.initials: Y
authors.lastname: Shang
authors.forename: Liang
authors.initials: L
authors.lastname: Li
authors.forename: Yongsu
authors.initials: Y
authors.lastname: Zhen
mesh_terms: D000230:Adenocarcinoma / Q000188:drug therapy; D000818:Animals; D000970:Antineoplastic Agents / Q000627:therapeutic use*; D001943:Breast Neoplasms / Q000188:drug therapy; D018826:CD13 Antigens / Q000378:metabolism*; D045744:Cell Line, Tumor; D053281:Enediynes / Q000627:therapeutic use*; D066246:ErbB Receptors / Q000235:genetics* / Q000276:immunology; D005260:Female; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000627:therapeutic use*; D057127:Single-Chain Antibodies / Q000235:genetics; D023041:Xenograft Model Antitumor Assays
chemical_list: D000970:Antineoplastic Agents; D053281:Enediynes; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D066246:ErbB Receptors; D018826:CD13 Antigens